垂体瘤转化基因1(PTTG1)的表达变化对人前列腺癌细胞LNCaP生长增殖的影响

垂体肿瘤转化基因1(PTTG1)具有促进肿瘤生长和转移的作用.通过上调或下调基因表达的策略,观察PTTG1基因对人前列腺癌细胞株LNCaP细胞生长增殖的影响.利用PCR技术分离出PTTG1全长cDNA,分别正向和反向插入真核表达载体pIRES2-EGFP,重组载体分别命名为正义PTTG1-S/pIRES2-EGFP(即pI-P-S)和反义PTTG1-AS/pIRES2-EGFP(即pI-P-AS),将这两种重组载体稳定转染LNCaP细胞,通过流式细胞仪和MTT法分别检测了细胞周期和细胞增殖的情况.转染正义PTTG1后处于S期和G2期的细胞明显增加,细胞生长增殖能力增强;相反,转染反义PTTG1后处于S期和G2期细胞明显减少,细胞生长增殖能力减弱(P<0.05).结果表明,PTTG1能明显改变人前列腺癌细胞株LNCaP的细胞周期和细胞生长增殖能力,它的异常表达可能参与前列腺癌细胞生长增殖过程.     

Generation of Mature Murine Monocytes from Heterogeneous Bone Marrow and Description of Their Properties

Monocytes are involved in a wide range of physiological and pathological processes, many of which are studied in mouse models. Current protocols to isolate murine monocytes are few and result in unsatisfactory cell yield and purity. Here, we describe a novel approach to efficiently differentiate large numbers of mature inflammatory monocytes from heterogeneous bone marrow cell suspensions. Bone marrow cell suspensions were isolated by flushing femurs and tibias from Balb/c and C57Bl/6 mice, supplemented with macrophage colony–stimulating factor (M-CSF), and were cultured on ultra-low attachment surfaces to inhibit adherence-mediated maturation. Cells were harvested at indicated time points, underwent time-line analysis of the differentiation processes, and were subsequently extensively phenotyped to verify their monocytotic properties. In order to confirm downstream compatibility, we tested for typical monocyte behavior. Our protocol yielded 24 ± 6 × 10⁶ differentiated cells per donor mouse, 10-fold higher than yields obtained using previously described peripheral blood isolation methods. Differentiated cells consisted of approximately 47% ± 12% monocytes, the rest being mature macrophages. We increased monocyte purity to 86% ± 6% by depleting adherent macrophages. Our findings indicate that bone marrow–derived monocytes (BMDMs) are an attractive tool to study, for example, the innate and adaptive immune system, atherosclerosis, and cellular migration during infection. Moreover, BMDM transplantation could be used to test novel, therapeutic in vivo approaches in mice disease models.     

含结构性倒位inv（5）（p13.1 q13.3）的毛细胞白血病细胞系的建立

为从分子水平揭示毛细胞白血病（HCL）的发病机理提供研究材料,将患者外周血淋巴细胞分离,用EBV病毒转化后进行细胞培养,建立细胞系,在连续传代3个月后,进行染色体组型分析,所培养细胞的染色体组型分析结果,与患者新鲜的外周血淋巴细胞的完全相同,证实该细胞系构建成功。     

HCMV感染对恶性胶质瘤U87细胞增殖及ATF5表达的影响

人巨细胞病毒(HCMV)能够诱导肿瘤细胞的恶性转化,但其分子机制尚有待进一步探索。探讨HCMV是否通过调控转录激活因子5(ATF5)的表达变化促进胶质瘤细胞的增殖。采用HCMV AD169株(MOI=5)感染神经胶质瘤U87细胞株,MTT方法观察HCMV感染0、12、24、48 h后细胞的增殖活性。Real-time PCR及Western-blot检测HCMV感染U87细胞后ATF5基因及蛋白的表达水平变化。以慢病毒为载体的靶向ATF5小干扰RNA构建载体,敲低ATF5表达水平后感染HCMV,MTT检测病毒感染细胞的增殖活性变化。HCMV感染神经胶质瘤U87细胞后,与未感染组比较,增值活性明显升高(P0.05),ATF5表达水平上升,表明HCMV感染使胶质瘤细胞增殖活性提高,细胞抗凋亡能力增强。成功构建沉默ATF5细胞系siATF5 U87,HCMV感染siATF5 U87细胞后使细胞增殖活性减弱,抗凋亡能力下降。以上实验结果表明,HCMV感染上调胶质瘤U87细胞ATF5的表达水平,促进细胞的增殖。因此HCMV感染可能通过调控ATF5信号通路增加细胞恶性性状,为治疗胶质瘤提供一个新的思路。     

绿豆根边缘细胞发育特性

以东北绿豆为试验材料,采用琼脂悬空培养法,研究了绿豆边缘细胞的发育特性。结果表明:绿豆根尖发育初期根边缘细胞呈球形,随着根尖伸长逐渐发育形成椭圆形、长椭圆形和长条形;发育过程中,根边缘细胞具有较高的存活率,在根长大于10mm后根边缘细胞的存活率均在70%～80%之间并趋于稳定;在根长为25～30mm时根边缘细胞数目达到最大值(约13 000个);根冠果胶甲基酯酶(PME)活性在根长5mm时达到最高值(1.486H+μmol.root cap-1.h-1),此后随着根的伸长,根冠PME活性在1.107～1.256H+μmol.root cap-1.h-1间变化并趋于稳定。     

基质金属蛋白酶MMP-26能有效促进人绒毛膜上皮癌细胞JEG-3浸润能力

胚胎植入过程中,滋养层细胞浸润与肿瘤的迁移过程非常相似,但显著的区别在于前者是受严格调控的有节制的浸润,基质金属蛋白酶(MMPs)的许多成员在其中起重要的作用.MMP-26是近年来发现的MMPs家族的新成员,它在滋养层细胞中的作用所知甚少.利用国际常用的人滋养层细胞模型——人绒毛膜上皮癌细胞系(JEG-3)作为体外实验模型,探讨MMP-26在人滋养层细胞浸润调节中的作用.将含有MMP-26全长cDNA的pCR3.1质粒转染到JEG-3细胞中,获得过量表达MMP-26基因的稳定细胞系JEG-3/MMP-26;细胞浸润分析表明JEG/MMP-26细胞的浸润能力较母本细胞明显增强;RT-PCR和明胶酶谱分析显示JEG-3/MMP-26细胞中MMP-9的表达和分泌水平提高;双荧光免疫细胞化学进一步显示MMP-26和MMP-9蛋白在细胞中有共定位现象.上述结果表明MMP-26能有效促进人滋养层细胞浸润,其作用可能是通过与其他MMP分子(如MMP-9)的协调来实现的.     

人肺腺癌细胞A—549和正常细胞HBE的蛋白质组差异分析

为了研究人肺腺癌细胞A 5 49和正常细胞HBE的蛋白质组差异 ,用固相pH梯度双向凝胶电泳分离人肺腺癌细胞系A 5 49和正常细胞HBE的总蛋白质 ,银染显色 ,PDQuest 2 DE软件分析 ,对部分差异蛋白质点进行基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)测定其胶内酶解后的肽质指纹图谱 ,用PeptIdent软件查询SWISS PROT数据库。结果获得了分辨率和重复性均较好的双向电泳银染图谱 ,图象分析探测到A 5 492 DE图谱的平均蛋白质点数为 (890± 38)个 ,HBE的平均蛋白质点数为 (75 7± 2 7)个 ,不同胶间蛋白质点的位置偏差在IEF方向为 (2 .85± 0 .48)mm ,在SDS PAGE方向为 (2 .6 9± 0 .37)mm。差异表达分析发现A 5 49和HBE图谱有5 35个蛋白质点相互匹配 ,其中A 5 49有 35 5个未被匹配 ,HBE中有 2 2 2个未被匹配 ;对A 5 49和HBE中的 18个差异蛋白质点分别进行肽质指纹分析 ,经数据库查询 ,初步鉴定为一些与物质代谢、细胞因子、信号转导有关的蛋白质。提示人肺腺癌细胞A 5 49和正常细胞HBE的蛋白质组具有差异 ,这种蛋白质组的差异分析有助于进一步研究肺腺癌的相关蛋白质及分子标记物     

绿色荧光蛋白标记的葡萄糖转运蛋白4稳定表达细胞系的建立

目的:建立稳定表达EGFP标记的葡萄糖转运蛋白4的CHO细胞系,为研究GLUT4在CHO细胞中的转运调节机制奠定基础。方法:采用分子克隆方法构建GLUT4-EGFP的融合蛋白,在FLP-in的CHO细胞系中表达,潮霉素筛选后得到稳定的细胞系。结果:通过共聚焦显微镜的检测,证明了此稳定细胞系的阳性率达到了99%。定位研究表明大部分GLUT4以囊泡形式分布在CHO细胞胞浆内,但是质膜上也有少量的GLUT4。结论:建立了一个稳定表达GLUT4-EGFP的CHO细胞系,为进一步研究GLUT4的转运提供了一个很好的细胞模型。     

The Purification and the Properties of Three Kinds of Lipases from Rhizopus delemer

The study was undertaken to clarify whether three kinds of lipases (EC 3.1.1.3) secreted from Rhizopus delemar are originally different or identical with each other. First of all, the purification of those lipases was carried out and their enzymatic properties were examined. Their properties including the stability on heat and pH, precipitabilities at a certain pH, the behaviours on a SE-Sephadex C50 column and on a Sephadex G200 column and so on were compared.

From the results, A-lipase is clearly different from the other two lipases. On the other hand, it seems that B- and C-lipases are originally identical.     

TF-1细胞凋亡相关基因的研究

利用近年来发展起来的代表差异分析（cDNA representational differences analysis, cDNA-RDA）技术研究了在人红白血病细胞株TF-1细胞撤除细胞因子后进入凋亡时诱导表达的基因.发现了6个新基因片段.其中有三个经与GenBank nr和dbEST查询均没有发现同源性,已经向GenBank进行登记,登记号分别为U83208,U83279,U83397.此外还发现一批已知基因的表达与凋亡相关,其中包括Hou和人硫氧还原蛋白等, 提示它们在凋亡中可能起作用.这项工作为进一步研究凋亡相关基因打下了良好基础.通过RDA的研究结果,有可能发现人白血病细胞凋亡的特异标记蛋白或发挥作用的重要蛋白,以期为白血病治疗提供理论基础.     

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the B...     

基于CRISPR/Cas9技术AEG-1基因敲除神经细胞系的构建及AEG-1基因敲除介导的神经细胞周期阻滞和细胞凋亡抑制

星形胶质细胞上调基因-1(AEG-1)是近年来研究较多的癌基因,但在神经系统疾病方面研究尚少。AEG-1与神经退行性疾病有关,然而其具体作用机制尚不明确。本研究通过设计靶向AEG-1 sgRNA序列并合成相应寡核苷酸,将其克隆到GV392质粒中,构建sgRNA/Cas9二合一表达载体,并进行慢病毒包装纯化。用慢病毒感染小鼠海马神经元HT22细胞,进行药物筛选和sgRNA活性鉴定,建立稳定的AEG-1基因敲除的细胞系;并进一步观察神经元HT22细胞的增殖与凋亡能力。结果显示,成功构建了3种靶向AEG-1基因的sgRNA/Cas9二合一表达载体。所设计的sgRNA的插入序列和开放阅读框架完全正确,成功建立了AEG-1基因敲除的稳转神经细胞系。进一步研究表明,AEG-1敲除后的神经HT22细胞与正常神经HT22细胞相比,细胞突起数目减少, 细胞周期阻滞,细胞凋亡率减少。以上结果为后续进一步研究AEG-1与神经系统疾病关系奠定了基础。     

Development of A MERS-CoV Replicon Cell Line for Antiviral Screening

Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease with a high mortality of ~ 35%. The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening. In this study, we constructed a MERS-CoV replicon containing the Renilla luciferase (Rluc) reporter gene and a stable luciferase replicon-carrying cell line. Using this cell line, we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir, indicating that this cell line can be used to screen inhibitors of MERS-CoV replication. Importantly, the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling, providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.     

干扰素膜蛋白Tet-on系统稳定细胞系的建立及对CA16病毒的抑制作用

通过Tet-on调控系统,构建受多西环素诱导表达干扰素诱导的跨膜蛋白(interferon-induced transmembrane proteins 1/2/3,IFITM1/2/3)基因的HeLa细胞系,并初步探索了IFITM蛋白对柯萨奇病毒A16(CA16)的抑制作用.首先将调控质粒pTet-on转染进入HeLa细胞,通过G418筛选出阳性克隆细胞系,在此细胞系基础上共同转染反应质粒pTRE2-IFITM1/2/3和伴侣质粒pTK-Hyg,通过潮霉素筛选出单克隆细胞系,加入多西环素后利用Western印迹筛选出可诱导表达IFITM1/2/3蛋白的单克隆细胞系.使用实时荧光定量PCR(RT-qPCR)检测发现,多西环素诱导表达的IFITM蛋白对不同感染复数(multiplicity of infection,MOI)的CA16具有明显的抑制作用,其中IFITM 3对CA16的抑制效果最为明显.Tet调控IFITM1/2/3基因表达HeLa细胞系的成功建立,为进一步研究IFITM基因的功能及其抗病毒机理提供了一个理想的细胞模型.     

Some Properties of Protease from Bacillus R-4 Which Lyses Rhizopus Cell Wall

A strain of Bacillus sp. (Bacillus R-4) produces a protease and a chitosanase which have the ability of lysing Rhizopus cell wall. Some enzymatic properties of the protease purified to a homogeneous state were examined.

The molecular weight of the enzyme was estimated as 19,000 and the isoelectric point as pH 8.65. The protease appeared to have a relative wide range of substrate specificity, hydrolyzing various proteins, such as gelatin, hemoglobin and protamine, and synthetic peptides, such as Z-Gly-Try-NH₂, Z-Gly-Ala-NH₂, Z-Ala-Leu-NH₂ and Z-Gly-Leu-NH₂. The activity lost by EDTA and by Hg²⁺ was restored by Zn²⁺ and reduced glutathione, respectively.     

聚乙二醇、葡聚糖对杂交瘤细胞保护特性研究

利用鼠鼠杂交瘤２Ｆ７细胞（分泌抗小细胞肺癌单克隆抗体）研究聚乙二醇、葡聚糖与杂交瘤细胞的生物相容性及添加限制浓度,研究添加浓度对葡萄糖消耗速率及氨形成速率的影响。在高速搅拌、高剪切力下考察了添加剂的保护性能。实验结果表明,０．０４％－０．１０％的聚乙二醇能较好地保护杂交瘤细胞,添加聚乙二醇后葡萄糖的比消耗速率增加,而氨的比生成速率没有变化;添加葡聚糖对葡萄糖的比消耗速率没有影响,但降低了氨的比生成速率,并且葡聚糖抑制细胞生长,不适合作动物细胞保护剂;小牛血清能较好地保护细胞;细胞死亡动力学表征为一级反应。在２５０ｍｌ磁力搅拌瓶中培养,培养基中添加０．１０％聚乙二醇,培养温度为３７．０,搅拌转速为８０转／分,细胞能止常生长;而培养基不添加聚乙二醇时细胞不能生长。     

细胞内F-actin的聚合与解聚对肝癌Bel-7402细胞的影响

目的：为探讨癌细胞内F—actin的解聚与聚合对癌细胞的形态、迁移、侵入的影响。方法：利用激光共聚焦显微镜对贴附培养的人肝癌：Bel-7402细胞形态及其细胞内F-actin进行观察;使用流式细胞仪对贴附的Bel一7402细胞及其脱落细胞与Cyt—B处理后Bel-7402细胞内F-actin的含量进行分析。结果：Bel-7402细胞在培养的过程中,癌细胞形态伸展,出现侵入性生长,细胞内F-actin聚合形成粗大的贯通细胞内的F-actin束,F-acfin含量增高;癌细胞在生长过程中,常出现重叠生长,细胞变圆,F_actin解聚变短,F-acfin小体增高,细胞有脱落的趋势,其脱落细胞内的F-actin含量低于贴附细胞。结论：人肝癌：Bel-7402细胞内F-actin的聚合可增加癌细胞的贴附和侵入性：细胞内F-actin的解聚,及Gactin重新聚合形成F-aefin小体可影响到癌细胞脱落及迁移。     

TTC-脱氢酶还原法测定螺旋藻细胞活性的条件优化

为确定氯化三苯基四氮唑(TTC)-脱氢酶还原法测定螺旋藻细胞活性的最优条件,首先通过单因素实验对影响藻细胞活性测定的TTC质量分数、缓冲液pH、提取剂（乙醇）质量分数、培养时间、培养温度进行分析,选定各因素的变化范围,再利用正交试验设计方法,在藻细胞活性测定的最适培养温度下,对TTC质量分数、缓冲液pH、提取剂质量分数、培养时间进行3水平优化试验。最后确定优化的TTC 脱氢酶还原法测定螺旋藻细胞活性的条件为TTC质量分数0.1%、缓冲液pH 8.0~8.5、乙醇质量分数60%、培养时间5 h、培养温度35 ℃。在此条件下所测藻细胞活性最高,为螺旋藻细胞活性的评价提供了一定参考。     

VEGF increases the proliferative capacity and eNOS/NO levels of endothelial progenitor cells through the calcineurin/NFAT signalling pathway

We have investigated whether VEGF (vascular endothelial growth factor) regulates the proliferative capacity and eNOS (endothelial nitric oxide synthase)/NO (nitric oxide) pathway of EPCs (endothelial progenitor cells) by activating CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) signalling. EPCs were obtained from cultured mononuclear cells isolated from the peripheral blood of healthy adults. Treatment with VEGF (50 ng/ml) potently promoted CaN enzymatic activity, activation of NFAT2, cell proliferation, eNOS protein expression and NO production. Pretreatment with cyclosporin A (10 μg/ml), a pharmacological inhibitor of CaN or 11R-VIVIT, a special inhibitor of NFAT, completely abrogated the aforementioned effects of VEGF treatment and increased apoptosis. The results indicate that VEGF treatment promotes the proliferative capacity of human EPCs by activating CaN/NFAT signalling leading to increased eNOS protein expression and NO production.